Microorganism markers are a necessary tool in the study, the manipulation, and the control of gene expression in the fields of research, production and control of microorganisms. This invention relates to the use of the lux AB gene cluster of Vibrio harveyi, a luminescent marine bacterium, as such a marker and/or tag.
Naturally occuring microorganisms need to be tagged or marked for monitoring their gene activity whenever that is necessary or desired for whatever reason. Moreover, ways need to be developed to label each genetically engineered organism so its use, identity and fate can be monitored in the field. This would greatly simplify studies of an organism's escape, survival, multiplication and dispersal and may help in tracking the movement of DNA among organisms in the environment.
The isolated lux AB gene cluster of V. harveyi was selected as a marker or tag because it was found to have several advantageous features including an unrestricted host background and an unrestricted growth medium requirement. In this respect, the lux AB genes of V. harveyi are different from the lac Z gene of Escherichia coli, a commonly used expression marker which, when fused with a promoter, has a restricted host background (i.e. it can be only used in microorganisms with no ability to utilize lactose). Most microorganisms will utilize lactose. Moreover, the cells containing lac Z fusions have to be grown in minimal media (a restricted number of components) in order for the lac Z product (B-galactosidase) to be detected. An especially attractive reason for using the lux AB gene of V. harveyi as a marker or tag for a microorganism is that it provides for the noninvasive measurement of the presence and absence of the marker through the presence of absence of bioluminescence as a measure of gene expression in vivo. The magnitude of bioluminescence is readily quantizible by new and known light detection techniques. The lac Z marker system does not have these capabilities. It uses a chemical substrate the color and intensity of which is the indicator of the reaction rate within the bacterium.
As known in the prior art, light is produced in E. coli cultures that contain seven lux genes cloned from another and different marine bacterium, Vibrio fischeri. (See an article entitled "Measuring Gene Expression with light" by Engebrecht, et al., Science, Volume 227, Mar. 15, 1985, pages 1345-1347). Therein it states in the abstract:
These lux genes were disconnected from their native promoter and inserted into the transposon mini-Mu. The resulting transposon, mini-Mu lux, could induce mutations by insertional inactivation of a target gene, and the lux DNA was oriented to align target gene transcription with that of the lux genes. Genes in Escherichia coli and Vibrio parahaemolyticus were mutagenized, and mutants containing transposon-generated lux gene fusions produced light as a function of target gene transcription. PA1 "The DNA encoding the luciferase alpha and beta subunits in the luminous marine bacterium Vibro harveyi (strain 392) is contained within a 4.0-kilobase HindIII fragment. DNA from V. harveyi was digested with HindIII, and the resulting fragments were inserted into the HindIII site of plasmid pBR322. The recombinant plasmids were introduced by transformation into Escherichia coli RRl. The colonies were supplied with n-decanal, the substrate for bioluminescence reaction, and 12 colonies (of ca. 6000 total) were observed to luminesce brightly. One of the recombinant plasmids, pTB7, has been studied in detail. The high level of expression of bioluminescence in pTB7 was the result not of native V. harveyi promoters but rather of a promoter in pBR322 which is within the tetracycline resistance gene but oriented in the direction opposite to the transcription of the tetracycline gene." PA1 1. The protection and identification of the modified microorganism. PA1 2. The detection of gene expression signals, including regulatory signals of the modified microorganism. PA1 3. The tracing of tumorous growth and cell migration by tracing a modified microorganism. PA1 4. The tracing of modified soil microorganisms and monitoring of their interaction with plants and other organisms.
However, the teachings of Engebrecht, et al. have limited application to the marking and tagging of microorganisms because all seven of the lux genes in V. fischeri are required for light production and the large size of this gene cluster increases the chance of its inactivation by mutation. Furthermore, it is not known whether the lux gene cluster of V. fischeri could reliably function as a measure of activity in microorganisms such as Bradyrhizobium. However, it does so function in E. coli and it does provide its own substrate for the production of light.
A new avenue for research and development in this field was presented by an article entitled "Cloning of the Luciferase Structural Genes from Vibrio harveyi and Expression of Bioluminescence in Escherichia coli" by Baldwin, et al., appearing in Biochemistry, 1984, Vol. 23, Pages 3663-3667. Therein bacterial luciferase in the luminous marine bacterium Vibrio harveyi was discussed as follows:
The recombinant plasmid vector pTB7 is available from the Agriculture Research Service Patent Collection, Northern Research Center, Peoria, Ill., 61604 and has been given the designation NRRL B-15231.
While the Baldwin et al. article describes the use of the lux AB genes of V. harveyi to select the recombinant plasmid vectors pTB7 in E. coli as stated, it is clear that it does not teach or show the isolation of those recombinant DNA vectors from the E. coli by any means for the purpose of establishing a marker or tag in E. coli or any other organism as a measure of gene expression. The recombinant plasmid vector pTB7 was isolated only for the purpose of cloning the luciferase gene lux AB. U.S. Pat. No. 4,581,335, inventor Thomas O. Baldwin, dated Apr. 8, 1986, which relates to the same research as the Baldwin et al. article, supplements the report by suggesting that the plasmid vector pTB7 be transferred to a host E. coli such as E. coli RRl. The transformed E. coli is given the designation E. coli/pTB7 and is deposited at the same place and under the same designation. The type of transformation involved is the transfer of the plasmids pTB 7 through and within the cell walls of the E. coli without insertion of the same into the chromosome of the E. coli. The plasmid pTB7 replicates outside of the chromosome of E. coli.